Extraction method for enhancing flavones in pericarpium citri reticulatae based on bio-transformation of lactobacillus casei

Abstract

The application discloses a pericarpium citri reticulatae flavone synergistic extraction method based on Lactobacillus casei bio-transformation, which comprises the following steps: a pericarpium citri reticulatae liquid preparation step, a Lactobacillus casei strain activation step, an inoculation and fermentation step and an extraction step. In the method, Lactobacillus casei is selected as a bio-transformation strain, and the Lactobacillus casei strain liquid is activated and fermented in the use process. The extraction rate of flavone extract can reach about 7%, which is significantly improved compared with a traditional extraction method. The content of active components such as flavone contained in the flavone extract can be increased by more than 4 times compared with the content of the traditional method. The anti-aging effect is tested by using a Caenorhabditis elegans anti-aging-aging pigment method, and it is found that, compared with a blank control group, the relative content of aging pigment of the tested sample is significantly reduced (P<0.05), and the removal rate of hydroxyl radicals is more than 69%.

Description

Technical Field

[0001] This invention relates to the chemical industry, and in particular to a method for enhancing the extraction of flavonoids from tangerine peel. Background Technology

[0002] Flavonoids are polyphenolic substances with a C6-C3-C6 structure, and they have wide applications in cosmetics, pharmaceuticals, food, and health products. Studies have found that dried tangerine peel is rich in flavonoids, and currently, the use of flavonoids on the market involves extracting flavonoid extracts from dried tangerine peel.

[0003] To obtain flavonoid extracts from dried tangerine peel, organic solvent extraction is currently the most common method, often with ultrasonic assistance. However, this method suffers from drawbacks such as complexity, low extraction rate (typically less than 3.5%), and residual organic solvents on the extract.

[0004] To address some of the aforementioned issues, microbial methods are now used to extract flavonoids from dried tangerine peel. However, this method suffers from insufficient strain specificity, resulting in a low extraction rate of flavonoids. Furthermore, the biotransformation efficiency of flavonoids is low, leading to a low concentration and instability of active ingredients such as flavonoids in the extract. Consequently, when these extracts are directly applied to cosmetics and other fields, they exhibit problems such as low transdermal absorption and unsatisfactory effects. Summary of the Invention

[0005] The purpose of this invention is to provide a method for extracting flavonoids from dried tangerine peel based on biotransformation of Lactobacillus casei, which can solve one or more of the above-mentioned problems.

[0006] According to one aspect of the present invention, a method for enhancing the extraction of flavonoids from dried tangerine peel based on Lactobacillus casei biotransformation is provided, comprising the following steps:

[0007] Preparation steps of tangerine peel liquid

[0008] The preparation steps of the tangerine peel liquid include taking tangerine peel, drying the tangerine peel and pulverizing it to obtain tangerine peel powder, adding the tangerine peel powder to boiled and cooled deionized water and stirring evenly to obtain tangerine peel liquid. The mass ratio of the tangerine peel to the deionized water is 1:30-50.

[0009] Activation steps for Lactobacillus casei strains

[0010] The activation step of the Lactobacillus casei strain includes taking a culture medium and a Lactobacillus casei culture solution. Each 200 ml of the culture medium contains 9 g to 10 g of MRS broth and 1.5 g to 2.5 g of agar. The culture medium is sterilized at high temperature. Then, 0.07 ml to 0.08 ml of Lactobacillus casei culture solution is inoculated onto the culture medium, with each 200 ml of the culture medium being inoculated. The culture medium is then activated to obtain an activated Lactobacillus casei culture solution.

[0011] Inoculation and fermentation steps,

[0012] The inoculation and fermentation step includes inoculating the activated Lactobacillus casei broth into the tangerine peel liquid, wherein the inoculation volume of the activated Lactobacillus casei broth to the tangerine peel liquid volume ratio is 0.05:280-520. The activated Lactobacillus casei broth is then fermented in the tangerine peel liquid to obtain a fermentation broth.

[0013] Extraction steps,

[0014] The extraction step includes drying the fermentation broth to obtain fermentation product, dissolving the fermentation product in 70% ethanol, wherein the mass of the 70% ethanol is 30 to 50 times the mass of the fermentation product, centrifuging to form a supernatant, and taking out the supernatant to obtain a flavonoid extract.

[0015] The beneficial effects of this invention are as follows: In this method, Lactobacillus casei is selected as the strain for biotransformation. During the process, the Lactobacillus casei culture is activated and fermented, and the final extraction rate of flavonoid extract can reach about 7%, which is significantly improved compared with traditional extraction methods. Furthermore, the content of active ingredients such as flavonoids in the flavonoid extract can be increased by more than 4 times compared with traditional methods. When the anti-aging effect of Caenorhabditis elegans was tested using the anti-aging pigmentation method, it was found that the test substance obtained by using this method had a significantly reduced relative content of aging pigments compared with the blank control group (P<0.05), and the hydroxyl radical removal rate reached more than 69%.

[0016] Meanwhile, in this method, the solvents used are mainly water and ethanol, which are easy to remove and do not easily remain on the flavonoid extract after removal, resulting in less environmental pollution and making it more green and environmentally friendly.

[0017] In some embodiments, during the preparation of the tangerine peel liquid, the tangerine peel powder can pass through a 100-mesh sieve. Through extensive experiments and testing, refining the tangerine peel powder to the above requirements allows for more effective extraction of its internal flavonoid-related substances.

[0018] In some embodiments, during the Lactobacillus casei activation step, the sterilization temperature of the culture medium is 120°C to 125°C, and the sterilization time is 13 min to 17 min. At this sterilization temperature and time, it is ensured that the bacteria in the culture medium are fully killed.

[0019] In some embodiments, during the activation step of the *Lactobacillus casei* strain, the activation temperature of the culture medium is 35°C to 40°C, and the activation time is 22 hours to 26 hours. At this activation temperature and time, the *Lactobacillus casei* in the culture medium can be effectively activated without harming it.

[0020] In some embodiments, during the inoculation and fermentation step, the fermentation temperature of the activated Lactobacillus casei culture in the tangerine peel liquid is 35°C to 40°C. At this fermentation temperature, effective fermentation of the activated Lactobacillus casei in the tangerine peel liquid can be ensured without harming it.

[0021] In some embodiments, during the inoculation and fermentation step, the fermentation time of the activated Lactobacillus casei culture in the tangerine peel liquid is 3 to 5 days. This fermentation time ensures that the activated Lactobacillus casei undergoes sufficient fermentation in the tangerine peel liquid.

[0022] In some embodiments, during the extraction step, the drying temperature is 50℃ to 70℃, and the drying time is 3 hours to 6 hours. This drying temperature and time effectively ensure the drying effect without damaging the active ingredients in the fermentation broth.

[0023] In some embodiments, during the extraction step, the centrifugation speed is 3800 r / min to 4200 r / min, and the centrifugation time is 20 min to 40 min. This centrifugation speed and time effectively ensure the centrifugation effect. Detailed Implementation

[0024] The present invention will now be described in further detail.

[0025] The present invention discloses a method for enhancing the extraction of flavonoids from dried tangerine peel based on Lactobacillus casei biotransformation, comprising the following steps:

[0026] a. Preparation steps of tangerine peel liquid.

[0027] The preparation steps of tangerine peel liquid include taking commercially available tangerine peel, washing it, drying it, and then pulverizing it using a pulverizer to obtain tangerine peel powder. The pulverized tangerine peel powder should be able to pass through a 100-mesh sieve, meaning that the diameter of each tangerine peel powder should be less than 150 micrometers.

[0028] The dried tangerine peel powder is added to boiled and cooled deionized water and stirred evenly to obtain tangerine peel liquid. The mass ratio of dried tangerine peel to deionized water should be 1:30-50. In this embodiment, it is preferable to take 10g of dried tangerine peel and 400g of deionized water, that is, take 400ml of deionized water.

[0029] b. Activation steps for Lactobacillus casei strains.

[0030] The activation steps of Lactobacillus casei include taking culture medium and Lactobacillus casei culture solution. Each 200ml of culture medium should contain 9g to 10g of MRS broth and 1.5g to 2.5g of agar. In this embodiment, it is preferable to take 200ml of culture medium containing 9.66g of MRS broth and 2g of agar.

[0031] The culture medium is sterilized by high temperature and high pressure. The sterilization temperature of the culture medium should be 120℃~125℃, and the sterilization time should be 13min~17min. In this embodiment, the sterilization temperature of the culture medium is preferably 121℃, and the sterilization time is preferably 15min.

[0032] Then, Lactobacillus casei culture was inoculated onto the culture medium using an inoculation loop. 0.07 ml to 0.08 ml of Lactobacillus casei culture was inoculated onto every 200 ml of culture medium. In this embodiment, it is preferable to inoculate 0.075 ml of Lactobacillus casei culture onto every 200 ml of culture medium.

[0033] The culture medium inoculated with Lactobacillus casei culture was then activated in an incubator to obtain activated Lactobacillus casei culture. The activation temperature of the culture medium should be 35℃~40℃, and the activation time should be 22h~26h. In this embodiment, the activation temperature of the culture medium is preferably 38℃, and the activation time is preferably 24h.

[0034] c. Inoculation and fermentation steps.

[0035] The inoculation and fermentation step includes inoculating activated Lactobacillus casei solution into tangerine peel solution using an inoculation loop. The inoculation volume ratio of activated Lactobacillus casei solution to tangerine peel solution should be 0.05:280-520. In this embodiment, the inoculation volume of activated Lactobacillus casei solution is preferably 0.05 ml, and the volume of tangerine peel solution is preferably 400 ml.

[0036] The activated Lactobacillus casei culture is then fermented in tangerine peel liquid to obtain a fermentation broth. The fermentation temperature of the activated Lactobacillus casei culture in the tangerine peel liquid should be 35℃~40℃, and the fermentation time should be 3 days~5 days. In this embodiment, the preferred fermentation temperature of the activated Lactobacillus casei culture in the tangerine peel liquid is 40℃, and the preferred fermentation time is 4 days.

[0037] d. Extraction steps.

[0038] The extraction step includes drying the fermentation broth at a temperature of 50°C to 70°C for 3 to 6 hours. In this embodiment, the drying temperature is preferably 60°C and the drying time is preferably 5 hours to obtain the fermentation product.

[0039] Take commercially available 70% ethanol (70% ethanol refers to an ethanol solution with a volume concentration of 70%), dissolve the fermentation product in the 70% ethanol, wherein the mass of the 70% ethanol should be 30 to 50 times the mass of the fermentation product. In this embodiment, the mass of the fermentation product is preferably 1g, and the mass of the 70% ethanol is preferably 40 times the mass of the fermentation product, that is, the mass of the 70% ethanol is preferably 40g.

[0040] The 70% ethanol containing the fermented material is centrifuged using a centrifuge. The centrifugation speed should be 3800 r / min to 4200 r / min, and the centrifugation time should be 20 min to 40 min. In this embodiment, the centrifugation speed is preferably 4000 r / min, and the centrifugation time is preferably 30 min. A supernatant is formed after centrifugation. The supernatant is taken out, and the supernatant is the flavonoid extract.

[0041] This method for enhancing the extraction of flavonoids from dried tangerine peel based on Lactobacillus casei biotransformation has the following advantages compared to traditional methods:

[0042] 1. Improved extraction efficiency: The final extraction rate of flavonoid extract reaches about 7%, which is significantly improved compared with traditional methods; the content of flavonoids and other active ingredients in the flavonoid extract obtained by this extraction method is more than 4 times higher than that of traditional methods.

[0043] 2. Safety optimization: The solvents used, such as deionized water and 70% ethanol, are easy to remove and leave little residue, effectively establishing a green extraction process with no solvent residue.

[0044] 3. Significant anti-aging effect: The anti-aging effect of Caenorhabditis elegans was tested using the anti-aging pigmentation method. It was found that the relative content of pigmentation in the test substance obtained by using this method was significantly reduced compared with the blank control group (P<0.05), and the hydroxyl radical removal rate reached more than 69%.

[0045] The above descriptions are merely some embodiments of the present invention. Those skilled in the art can make various modifications and improvements without departing from the inventive concept of the present invention, and these all fall within the scope of protection of the present invention.

Claims

1. A method for enhancing the extraction of flavonoids from tangerine peel based on Lactobacillus casei biotransformation, characterized in that, Includes the following steps: Preparation steps of tangerine peel liquid The preparation steps of the tangerine peel liquid include taking tangerine peel, drying the tangerine peel and pulverizing it to obtain tangerine peel powder, adding the tangerine peel powder to boiled and cooled deionized water and stirring evenly to obtain tangerine peel liquid. The mass ratio of the tangerine peel to the deionized water is 1:30-50. Activation steps for Lactobacillus casei strains The activation step of the Lactobacillus casei strain includes taking a culture medium and a Lactobacillus casei culture solution. Each 200 ml of the culture medium contains 9 g to 10 g of MRS broth and 1.5 g to 2.5 g of agar. The culture medium is sterilized at high temperature. Then, 0.07 ml to 0.08 ml of Lactobacillus casei culture solution is inoculated onto the culture medium, with each 200 ml of the culture medium being inoculated. The culture medium is then activated to obtain an activated Lactobacillus casei culture solution. Inoculation and fermentation steps, The inoculation and fermentation step includes inoculating the activated Lactobacillus casei broth into the tangerine peel liquid, wherein the inoculation volume of the activated Lactobacillus casei broth to the tangerine peel liquid volume ratio is 0.05:280-520. The activated Lactobacillus casei broth is then fermented in the tangerine peel liquid to obtain a fermentation broth. Extraction steps, The extraction step includes drying the fermentation broth to obtain fermentation product, dissolving the fermentation product in 70% ethanol, wherein the mass of the 70% ethanol is 30 to 50 times the mass of the fermentation product, centrifuging to form a supernatant, and taking out the supernatant to obtain a flavonoid extract.

2. The method for enhancing the extraction of flavonoids from tangerine peel based on Lactobacillus casei biotransformation according to claim 1, characterized in that, In the preparation steps of the tangerine peel liquid, the tangerine peel powder can pass through a 100-mesh sieve.

3. The method for enhancing the extraction of flavonoids from tangerine peel based on Lactobacillus casei biotransformation according to claim 1, characterized in that, In the activation step of the Lactobacillus casei strain, the sterilization temperature of the culture medium is 120℃~125℃, and the sterilization time of the culture medium is 13min~17min.

4. The method for enhancing the extraction of flavonoids from tangerine peel based on Lactobacillus casei biotransformation according to claim 1, characterized in that, In the activation step of the Lactobacillus casei strain, the activation temperature of the culture medium is 35℃~40℃, and the activation time of the culture medium is 22h~26h.

5. The method for enhancing the extraction of flavonoids from tangerine peel based on Lactobacillus casei biotransformation according to claim 1, characterized in that, In the inoculation and fermentation step, the fermentation temperature of the activated Lactobacillus casei culture in the tangerine peel liquid is 35℃~40℃.

6. The method for enhancing the extraction of flavonoids from tangerine peel based on Lactobacillus casei biotransformation according to claim 5, characterized in that, In the inoculation and fermentation step, the fermentation time of the activated Lactobacillus casei culture in the tangerine peel liquid is 3 to 5 days.

7. The method for enhancing the extraction of flavonoids from tangerine peel based on Lactobacillus casei biotransformation according to claim 1, characterized in that, In the extraction step, the drying temperature is 50℃~70℃, and the drying time is 3h~6h.

8. The method for enhancing the extraction of flavonoids from tangerine peel based on Lactobacillus casei biotransformation according to claim 1, characterized in that, In the extraction step, the centrifugation speed is 3800 r / min to 4200 r / min, and the centrifugation time is 20 min to 40 min.